The long-term goal of this project is to understand how HIV-1 Net pirates the cellular protein trafficking and signal transduction machinery to counterattack the host cell antiviral response. Host cells have a two-armed response to combat viruses: immune surveillance and apoptosis. HIV-1 uses primarily a single gene, Nef, to block the multiple aspects of this response. For example, Nef induces MHC-I downregulation, a block of apoptosis, and disruption of T-cell development in the thymus. How Nef plays so many different roles in the counterattack of the antiviral response remains unclear. We have identified the underlying membrane trafficking/signaling pathway used by HIV-1 Nef to downregulate MHC-I. We determined that Nef and the cellular trafficking protein PACS-1 combine to usurp a PI3K-dependent, ARF6-controlled endocytic pathway to downreg ulate cell-surface MHC-I to the trans-Golgi network. This discovery suggests that the PACS- 1/Nef controlled activation of PI3K may be important for downregulation of MHC-I and also for implementing the entire array of HIV-1 Nef's counterattack measures. Our preliminary data suggests a novel signaling pathway is the key to this implementation, but much about this pathway remains unknown. We have found that this pathway begins when HIV-1 Nef pirates PACS-1 to target to the TGN, enabling Nef to recruit a Src family kinase (SFK); subsequently, this complex binds to and activates PI3K. PI3K then activates ARF6 to downregulate MHC-I to the TGN. This pathway also activates PAK2, which blocks apoptosis and appears to be required for HIV-1 to arrest T-cell maturation. Our central hypothesis is that this PACS-1/Nef-SFK-PI3K pathway allows Nef to thwart the host's antiviral response. Aim 1 will determine how HIV-1 Nef usurps the PACS-1 sorting pathway to recruit SFKs at the TGN. Aim 2 will determine how HIV-1 Nef subverts SFKs to activate PI3K and will test if the PACS-1/Nef-SFK-PI3K axis coordinates the counterattack of the host antiviral response in different cellular reservoirs. Aim 3 will determine how HIV-1 Nef pirates the ARF6- controlled endocytic pathway to downregulate MHC-I to the TGN. Elucidating how this Nef-induced signaling pathway is used by HIV-1 will provide better understanding of the biochemical basis underlying HIV-1 pathogenesis and will identify new targets to combat this deadly virus.